Active modified hedgehog proteins

ABSTRACT

A post-translationally processed hedgehog protein mutant which  
     exhibits a molecular weight of 22±1 kDa under alkylating conditions,  
     exhibits a molecular weight of 24±1 kD under reducing conditions,  
     is stabilized with respect to its activity by suramin  
     is inactivated when 8 or more amino acids are cleaved N-terminally  
     is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37° C.,  
     induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,  
     is not modified by cholesterol,  
     exhibits an activity that is increased many times.

[0001] The invention concerns an active form of a hedgehog protein, aprocess for its recombinant production and its therapeutic use. Hedgehog(hh) proteins are understood as a family of secreted signal proteinswhich are responsible for the formation of numerous structures inembryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80(1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood etal., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996)613, C. J. Lai et al., Development 121 (1995) 2349). During itsbiosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain areobtained after cleavage of the signal sequence and autocatalyticcleavage. The N-terminal fragment is modified with lipid (J. A. Porteret al., Science 274 (1996) 255-259). In higher life-forms the hh familyis composed of at least three members i.e. sonic, indian and desert hh(Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51).Differences in the activity of hedgehog proteins that were producedrecombinantly were observed after production in prokaryotes andeukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura etal., Biochem. Biophys. Res. Comm. 237 (1997) 465-469.

[0002] Hynes et al. compare the activity of hh in the supernatant oftransformed human embryonic kidney 293 cells (eukaryotic hh) with hhproduced from E. coli and find a four-fold higher activity of hh fromthe supernatants of the kidney cell line. A potential additionalaccessory factor which is only expressed in eukaryotic cells, apost-translational modification, a different N-terminus since the hhisolated from E. coli contains 50% of a hh which carries two additionalN-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, ora higher state of aggregation (e.g. by binding to nickel agarose beads)have been discussed to be the reason for this increased activity.

[0003] Nakamura et al. compare the activity of shh in the supernatant oftransformed chicken embryo fibroblasts with an shh fusion proteinisolated from E. coli which still has an N-terminal polyhistidine part.The shh in the supernatant of the fibroblasts has a seven-fold higheractivity than the purified E. coli protein with regard to stimulation ofalkaline phosphatase (AP) in C3H10T ½ cells. Molecules such as bonemorphogenetic proteins (BMPs) have been discussed as the reason for theincreased activity which are only present in the supernatant ofeukaryotic cells and cause the stronger induction of AP.

[0004] Kinto et al., FEBS Letters, 404 (1997) 319-323 describe thatfibroblasts which secrete hh induce ectopic bone formation in an i.m.implantation on collagen.

[0005] The object of the invention is to produce hh proteins(polypeptide) which have a considerably improved activity compared tothe known forms.

[0006] The object is achieved by a post-translationally processedhedgehog protein mutant (hh mutant) which is obtainable by expression ofa gene which codes a hedgehog protein in a baculovirus expression systemin a fermentation over a period of up to 30 hours, preferably 24-27hours, purification of the cell supernatant in the presence of aprotease inhibitor and a non-ionic detergent and isolation of the hhmutant which binds to heparin-Sepharose and hydroxylapatite and ischaracterized in that this hh mutant

[0007] exhibits a molecular weight of 22+1 kDa under alkylatingconditions,

[0008] exhibits a molecular weight of 24+1 kD under reducing conditions,

[0009] is stabilized with respect to its activity by suramin

[0010] is inactivated when 8 or more amino acids are cleavedN-terminally

[0011] is inactivated by 90% or more when incubated with 10 mmol/l 1.4dithioerythritol (DTE) for 2.5 hours at 37° C.,

[0012] induces an activity for alkaline phosphatase of ca. 90 nmolpNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,

[0013] is not modified by cholesterol.

[0014] Activity within the sense of the invention is understood as theactivity of alkaline phosphatase which the polypeptide can induce inmammalian cells (activity in the alkaline phosphatase test). In thismethod a mouse fibroblast cell line is cultured in a medium whichcontains foetal calf serum. Subsequently sterile filtered sample isadded, the cells are lysed after ca. 5 days and alkaline phosphatase isdetermined in the cell lysate by means of the cleavage of a chromogenicsubstrate (pNP, p-nitrophenol) (J. Asahina, Exp. Cell. Res. 222 (1996)38-47 and T. Nakamura (1997)).

[0015] A baculovirus expression system is understood as an expressionsystem composed of a baculovirus vector and an insect cell as the hostcell. Such expression systems are known to a person skilled in the artand are for example described by Bumcrot (1995) for hh proteins.

[0016] A hedgehog protein is understood by the invention as a secretedsignal protein which is responsible for the formation of numerousstructures in embryogenesis. Sonic, indian or desert hh are particularlypreferably used (M. Fietz et al. (1994). A hh protein with a sequence asdescribed in the EMBL database under the No. L38518 is preferably used.Proteins of the hedgehog family exhibit a pronounced homology in theiramino acid sequence which is why it is also preferable to express thosenucleic acids which code for hedgehog proteins which are 80% or morehomologous with the above-mentioned sequence of sonic hedgehog protein.

[0017] The sonic hedgehog precursor protein is composed of the aminoacids 1-462 of the sequence described in the EMBL database under No.L38518. The amino acids 1-23 represent the signal peptide, the aminoacids 24-197 represent the mature signal domain, the amino acids 32-197represent the signal domain shortened by 8 amino acids and the aminoacids 198-462 represent the auto-processing domain after autoproteolyticcleavage.

[0018] The first 8 amino acids of the hedgehog protein are understood bythe invention as the first 8 amino acids of the processed protein forexample the amino acids 24-31 for sonic hedgehog protein.

[0019] Surprisingly when hedgehog proteins are produced recombinantly inthe baculovirus expression system, a highly active mutant of the protein(activity increased by at least 10-fold, preferably at least 100-foldcompared to shh) accumulates in the initial period of the fermentation.This mutant of the polypeptide according to the invention can be inparticular isolated when the fermentation is terminated at the latestafter ca. 30 hours, preferably after ca. 24-27 hours. This is alsosurprising since a fermentation period after infection of at least 2days has been previously described for the production of hh proteins inthe baculovirus expression system (Bumcrot et al., Mol. Cell. Biol.(1995) 2294-2303). It has also been described for other proteins whichare produced in the baculovirus system such as rhodopsin kinase (Cha etal., Proc. Natl. Acad. Sci. USA 94 (1997) 10577-10582) that a maximum ofprotein and activity is achieved after 64-88 h. According to theinvention it was found for hedgehog proteins that although the amount ofhedgehog protein in the fermentation supernatant greatly increases inthe period between 33 and 72 hours, mainly hh protein with an activitythat is known from the prior art is formed in this period. In contrastthe amount of such a hh protein is considerably less (at least5-10-fold) when the fermentation period is reduced to below ca. 30 h.which allows the identification and isolation of the highly active hhprotein mutant according to the invention.

[0020] The hh mutant according to the invention is very sensitivetowards proteases which is why it is preferable to add proteaseinhibitors such as for example aprotinin, PMSF or pepstatin or a mixturethereof to the supernatant of the fermentation. Furthermore it ispreferable to add non-ionic detergents such as polysorbate (e.g.Triton®X100) during the purification, preferably at least after thefirst crude purification over heparin-Sepharose, since this alsostabilizes the hh proteins according to the invention.

[0021] In a first step for the purification of the protein according tothe invention it is expedient to carry out a chromatography onheparin-Sepharose. It is preferable to carry out this chromatography asa step elution i.e. preferably to elute at a concentration of at least0.7 mol/l (preferably 1.2 mol/l) after washing with 250 mmol/l NaCl.

[0022] It is particularly preferable to carry out a hydroxylapatitechromatography to purify the hh mutant according to the invention. Thisachieves a good concentration of the activity with relatively low losses(<50%). Further suitable chromatographic steps are for example aheparin-Sepharose chromatography (Miao et al., J. Neurosci. 17 (1997)5891-5899) which is, however, preferably carried out in the presence ofnon-ionic detergents. Furthermore it is preferable to carry out adialysis after the heparin-Sepharose chromatography preferably againstlow ionic strengths (e.g. buffer containing 1-10 mmol/l sodium phosphatepH 6.5-7.5).

[0023] It is particularly preferable that in this dialysis the bufferagainst which it is dialysed contains 10-100 mmol/l, preferably 50mmol/l sodium chloride and that the dialysis is carried out at a lowconcentration of the hh protein (1 mg/ml or less, preferably 0.5 mg/mlor less).

[0024] Furthermore it is also preferable to add suramin during thepurification or at least before determining the activity of the protein.This also stabilizes the activity. In the case of suramin it waspreviously only known that it is suitable for detaching hh proteins fromthe cell surface or the extracellular matrix (Bumcrot et al., seeabove).

[0025] For the further purification it is preferable to againchromatograph on heparin-Sepharose and hydroxylapatite.

[0026] In a further embodiment of the invention the hh mutant accordingto the invention can be used to produce a pharmaceutical compositionwhich is also a subject matter of the invention. This pharmaceuticalcomposition contains a pharmacologically effective dose of the proteinaccording to the invention and can be administered systemically as wellas locally. It is also preferable to use the proteins according to theinvention in combination with other proteins of the hedgehog family orbone growth factors such as bone morphogenetic proteins (BMPs), (Wozneyet al., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and theirGene Expression, 131-167, Academic Press Inc. 1993) or parathyroidhormones (Karablis et al., Genes and Development 8 (1994) 277-289).

[0027] The protein according to the invention can be used advantageouslyto induce chondrocytes and osteocytes in an osteoinductivepharmaceutical composition. Osteoinductive pharmaceutical compositionsare for example known from the U.S. Pat. No. 5,364,839, WO 97/35607, WO95/16035.

[0028] When the protein according to the invention is administeredlocally it is preferable to use it in combination with a suitable matrixas a carrier and/or with a sequestering agent. Such a matrix is suitablefor slowly releasing the protein in vivo in an active form in particularin the vicinity of bones. The sequestering agent is a substance whichfacilitates administration for example by injection and/or prevents orat least delays migration of the protein according to the invention fromthe site of administration.

[0029] A biocompatible degradable material for example based on collagenor other polymers based on polylactic acid, polyglycolic acid orco-polymers of lactic acid and glycolic acid are particularly suitableas a matrix material. Such polymer matrices are described for example inWO 93/00050.

[0030] Sequestering agents are for example cellulose and cellulose-likematerials and for example alkyl cellulose, carboxymethyl cellulose,hyaluronic acid, sodium alginate, polyethylene glycol and polyvenylalcohol of which hyaluronic acid is particularly preferred especially ina pharmaceutical composition even without carrier matrix.

[0031] It is also preferable for the production of the pharmaceuticalcomposition to add auxiliary substances such as mannitol, sucrose,lactose, glucose or glycine and antioxidants such as EDTA, citrate anddetergents, preferably non-ionic detergents like polysorbates andpolyoxyethylenes.

[0032] In a further preferred embodiment a pharmaceutical composition ofthe hedgehog protein according to the invention together with suramin ispreferred and can be advantageously used.

[0033] The following examples, publications and figures furtherelucidate the invention, the protective scope of which results from thepatent claims. The described methods are to be understood as exampleswhich still describe the subject matter of the invention even aftermodifications.

DESCRIPTION OF THE FIGURES

[0034]FIG. 1: Kinetics of the secretion of alkaline phosphatase inducingactivity (bars) and hh mutant (dots and line) by high five cells afterinfection with baculovirus.

[0035]FIG. 2: Elution diagram of the purification of the fermentationsupernatant with heparin-Sepharose

[0036]FIG. 3: Elution diagram of the purification of the dialysed eluateof the heparin-Sepharose with hydroxylapatite

[0037]FIG. 4: Elution diagram of the purification of the dialysed activefractions of the hydroxylapatite column with a 1 ml HiTrap heparincolumn.

[0038]FIG. 5: Alkaline phosphatase inducing activity of the fractions ofthe 1 ml High Trap heparin chromatography.

[0039]FIG. 6: Coomassie staining of SDS-PAGE with alkylated fractions ofthe 1 ml High Trap heparin chromatography.

[0040]FIG. 7: Western blot with an antibody against the N-terminus ofshh of the SDS-PAGE with alkylated samples of the fractions of the 1 mlHigh Trap heparin chromatography.

[0041]FIG. 8: Western blot with an antibody against the N-terminus ofshh of the SDS-PAGE with reduced samples of the fractions of the 1 mlHigh Trap heparin chromatography.

[0042]FIG. 9: Activity of the retained and permeated material afterfiltration of active shh fractions using membranes with an exclusionlimit of 30 and 100 KDa

[0043]FIG. 10: Influence of suramin on the activity of the hh mutant: Nosuramin (B), suramin ad 0.1 mg/ml only added after dialysis againstPBS+0.05% Tween®80 (C) or suramin ad 0.1 mg/ml added before dialysis anddialysed against PBS+0.05% Tween®80 containing additionally 0.1 mg/mlsuramin were added to aliquots of an active fraction afterhydroxylapatite chromatography.

[0044]FIG. 11: Influence of Tween®20 and Tween®80 on the activity of thehh mutant: Aliquots of a pool of AP active fractions after SP Sepharosechromatography in 50 mM NaPi, 0.9 M NaCl, 1 mM EDTA pH 7.3 were admixedwith the stated concentrations of Tween and dialysed against PBScontaining the respective concentration of Tween. The samples weresterile filtered through 0.2 μm filters before being used in the C3H10T½test.

[0045]FIG. 12: Influence of trypsin and chymotrypsin on the activity ofthe hh mutant: AP active fractions after a step elution ofheparin-Sepharose were adjusted to a protein concentration of 0.46 mg/mlin 10 mM Na phosphate, 0.05% Tween®80 and admixed with trypsin orchymotrypsin at a protease/protein ratio (w/w) of 1:100 (A), 1:500 (B),1:2500 (C) and 1:10000 (D). The samples were incubated for 11 h at RT.The digestion was stopped by adding aprotinin in a 5-fold weight excessand the samples were analysed in SDS-PAGE (A:) and in the C3H10T½ test(B:). 1, test mixture; 2, control without protease; 3, samples treatedwith trypsin; 4, samples treated with chymotrypsin; 5, control trypsin(1:100) and aprotinin at t=0; 6, control chymotrypsin (1:100) andaprotinin at t=0.

EXAMPLE 1

[0046] Expression of Recombinant Human Sonic hh (shh)

[0047] The N-terminal domain of human shh with the amino acids 24-197(EMBL accession No. L 38518) was as described by Miao (J. Neurosci.(1997) 17, 5891-5899) and Bumcrot et al., (Mol. Cell. Biol. (1995) 15,2294-2303) for the rat protein by means of recombinant baculovirus inHigh five cells (Invitrogen, Leek, NL, Order No. E 855-O₂) using Excell400 medium (JHR, Inc.) in which sufficient virus was used to infect eachcell on average with one virus (multiplicity of infection (m.o.i.):1).

[0048] The fermenter contents were clarified after 26 or 72 h bycentrifugation at 1000 g and filtration and the supernatant or thepermeate was stored at −80° C. until further use. Fermentation sampleswere analysed for their content of alkaline phosphatase inducingactivity [Nakamura et al. (1997), Kinto et al. (1997) FEBS Lett. 404,319-323] and for their content of shh protein by means of RP-HPLC (VydacC18, gradient of 0-90% acetonitrile in 0.1% trifluoroacetic acid, TFA)or SDS-PAGE.

[0049] The fermentation was terminated after 24-32 h (preferably after24-27 h) fermentation time and the supernatant was clarified.

EXAMPLE 2

[0050] Purification of the Active hh Mutant

[0051] 1 Tablet of “complete” inhibitor mix (Boehringer Mannheim GmbH,order No. 1873580) was added per 50 ml supernatant to the clarifiedsupernatant after thawing and 3.5 l of this solution was applied at 4°C. to a heparin-Sepharose column (volume 90 ml; Pharmacia Biotech) whichhad previously been equilibrated with 20 mM sodium phosphate, pH 7.2.After the sample application it was washed with 20 mM sodium phosphate,0.05% Tween®80, pH 7.2 (=buffer A) and unspecifically bound protein waseluted by a wash step with buffer A which additionally contained 0.25 MNaCl. The activity was obtained by a subsequent elution with buffer Awhich additionally contained 1.2 M NaCl.

[0052] This eluate was subsequently diluted with one volume 10 mM sodiumphosphate, 0.05% Tween®80, 50 mM NaCl, pH 7.2 (=buffer B) and dialysedagainst buffer B at 4° C.

[0053] The dialysate was applied to a hydroxyapatite column (volume 10ml; Makro Prep; 40 μm, type I; BIO-Rad) equilibrated with buffer B. Itwas eluted with a gradient of 10 to. 300 mM NaP in buffer B (2×200 ml).

[0054] Aliquots of the fractions were analysed for their ability tostimulate alkaline phosphatase in a mouse fibroblast cell line e.g.C3H10T½ cells as well as by means of SDS-PAGE and RP-HPLC. The remainderof the fractions was stored at −80° C. until further processing. Themaximum activity elutes at the end of the gradient between 0.25-0.3 Msodium phosphate whereas inactive or only weakly active forms of shhalready elute much earlier from the column.

[0055] The active fractions were pooled and dialysed against buffer B at−4° C. and applied to a 1 ml HiTrap heparin column (Pharmacia Biotech)which had been equilibrated with 20 mM potassium phosphate, 0.05Tween®80, pH 7.2. It was eluted by a gradient of 0-1400 mM KCl in 20 mMpotassium phosphate, 0.05% Tween®80, 50 mM NaCl, pH 7.2. Activefractions were identified by the stimulation of alkaline phosphatase inC3H10T½ cells, and alkylated and reduced samples were analysed by meansof SDS-PAGE and Western blot with an antibody against the N-terminus ofshh.

[0056] In contrast to inactive fractions, the active fractions contain ahh mutant which under alkylating conditions exhibits a molecular weightwhich is 1-3 kDa lower than only weakly active shh with an intact Nterminus. However such a difference in molecular weight is notdetectable under reducing conditions.

EXAMPLE 3

[0057] Induction of Alkaline Phosphatase in the Cell Test (Determinationof the Activity of Alkaline Phosphatase)

[0058] 5000 cells of the murine mesenchymal pluripotent line C3H10T½(ATCC CCL-226) were sown in each well of a 96-well microtitre plate. Thecells were in 100 μl DMEM, 2 mM glutamine, 100 IU/ml penicillin, 100μg/ml streptomycin and 10% foetal calf serum, FCS. On the next day theactive substances to be examined were added at the appropriateconcentrations in a volume of 100 μl. The test was stopped after 5 days.For this purpose the supernatants were discarded and the cells werewashed once with PBS. The cells were lysed in 50 μl 0.1% Triton®X-100and frozen at −20° C. After thawing 25 μl was used for the proteindetermination and 25 Al for the determination of the activity ofalkaline phosphatase.

[0059] Protein Determination According to the Instructions of theManufacturer Pierce:

[0060] 75 μl redistilled H₂O was added to the mixture, then 100 μl BCAprotein reagent was added (Pierce Micro BCA, No. 23225). After 60 minthe optical density (OD) at 550 nm was measured.

[0061] Activity of the Alkaline Phosphatase According to theInstructions of the Manufacturer Sigma:

[0062] 100 μl reaction buffer (Sigma 221) was added to the preparation.A substrate capsule (Sigma 104-40) was dissolved in 10 ml redistilledH₂O and then 100 μl was added to the test mixture by pipette. The OD wasmeasured at 405 nm after the yellow coloration. In the reaction alkalinephosphatase converts p-nitrophenyl phosphate into p-nitrophenol.

[0063] The ODs were each converted into nmol or μg by means of standardcurves. The evaluation was according to the formula:

[0064] nmol PNP per (measured) minute per mg (cell) protein

[0065] Results:

[0066] Shh (monomer) from E. coli at a concentration of 43 μg/ml (=2.15μmol/l) in the cell test results in 8.537 nmol pNP/min/mg

[0067] Shh (dimer) from E. coli at a concentration of 41.5 μg/ml (=1.037pmol/1) in the cell test results in 5.133 nmol pNP/min/mg

[0068] hh mutant according to the invention at a concentration of 0.1μg/ml (=5 nmol/1) with 0.1 mg/ml suramin in the cell test results in88.762 nmol pNP/min/mg

[0069] hh mutant according to the invention at a concentration of 0.1μg/ml (=5 nmol/1) without suramin in the cell test results in 44.828nmol pNP/min/mg

[0070] a control without shh results in 1.292 nmol pNP/min/mg

[0071] BVCM used in a 1/40 dilution results in 41.961 nmol pNP/min/mg(=internal positive control)

LIST OF REFERENCES

[0072] Asahina, J., Exp. Cell. Res. 222 (1996) 38-47

[0073] Bitgood, M. J. et al., Curr. Biol. 6 (1996) 296

[0074] Bumcrot et al., Mol. Cell. Biol. (1995) 2294-2303

[0075] Cha et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10577

[0076] Chiang, C. et al., Nature 83 (1996) 407

[0077] Fietz, M. et al., Development (Suppl) (1994) 43-51

[0078] Hynes, M. et al., Neuron 15 (1995) 35-44

[0079] Karablis et al., Genes and Development 8 (1994) 277-289

[0080] Kinto et al., FEBS Letters, 404 (1997) 319-323

[0081] Lai, C. J. et al., Development 121 (1995) 2349

[0082] Miao et al., J. Neurosci. 17 (1997) 5891-5899

[0083] Nakamura, T. et al., Biochem. Biophys. Res. Comm. 237 (1997)465-469

[0084] Perrimon, N., Cell 80 (1995) 517-520

[0085] Porter, J. A. et al., Science 274 (1996) 255-259

[0086] Smith, J. C., Cell 76 (1994) 193-196

[0087] U.S. Pat. No. 5,364,839

[0088] Vortkamp, A. et al., Science 273 (1996) 613

[0089] WO 97/35607

[0090] WO 93/00050

[0091] WO 95/16035

[0092] Wozney et al., Cell. Mol. Biol. of Bone, Bone MorphogeneticProteins and their Gene Expression, 131-167, Academic Press Inc. 1993

1 1 1 462 PRT Homo sapiens Human Sonic hedgehog 1 Met Leu Leu Leu AlaArg Cys Leu Leu Leu Val Leu Val Ser Ser Leu 1 5 10 15 Leu Val Cys SerGly Leu Ala Cys Gly Pro Gly Arg Gly Phe Gly Lys 20 25 30 Arg Arg His ProLys Lys Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile 35 40 45 Pro Asn Val AlaGlu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60 Lys Ile Ser ArgAsn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80 Asn Pro AspIle Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95 Leu Met ThrGln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser 100 105 110 Val MetAsn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp 115 120 125 AspGlu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg 130 135 140Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly Met 145 150155 160 Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu165 170 175 Ser Lys Ala His Ile His Cys Ser Val Lys Ala Glu Asn Ser ValAla 180 185 190 Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val HisLeu Glu 195 200 205 Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Ser Pro GlyAsp Arg Val 210 215 220 Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr SerAsp Phe Leu Thr 225 230 235 240 Phe Leu Asp Arg Asp Asp Gly Ala Lys LysVal Phe Tyr Val Ile Glu 245 250 255 Thr Arg Glu Pro Arg Glu Arg Leu LeuLeu Thr Ala Ala His Leu Leu 260 265 270 Phe Val Ala Pro His Asn Asp SerAla Thr Gly Glu Pro Glu Ala Ser 275 280 285 Ser Gly Ser Gly Pro Pro SerGly Gly Ala Leu Gly Pro Arg Ala Leu 290 295 300 Phe Ala Ser Arg Val ArgPro Gly Gln Arg Val Tyr Val Val Ala Glu 305 310 315 320 Arg Asp Gly AspArg Arg Leu Leu Pro Ala Ala Val His Ser Val Thr 325 330 335 Leu Ser GluGlu Ala Ala Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly 340 345 350 Thr IleLeu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val Ile Glu 355 360 365 GluHis Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu Ala His 370 375 380Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg Gly Gly Asp 385 390395 400 Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gly Arg Val Ala Leu Thr405 410 415 Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala Gly Ala Thr Ala GlyIle 420 425 430 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gly Thr Trp LeuLeu Asp 435 440 445 Ser Glu Ala Leu His Pro Leu Gly Met Ala Val Lys SerSer 450 455 460

1. Post-translationally processed hedgehog protein mutant which isobtainable by expressing a gene which codes for a hedgehog protein in abaculovirus expression system in a fermentation for a period of up to 30hours, purifying the cell supernatant in the presence of a proteaseinhibitor and a non-ionic detergent and isolating the hh mutant whichbinds to heparin-Sepharose and hydroxylapatite and is characterized inthat this hh mutant exhibits a molecular weight of 22+1 kDa underalkylating conditions, exhibits a molecular weight of 24+1 kD underreducing conditions, is stabilized with respect to its activity bysuramin is inactivated when 8 or more amino acids are cleavedN-terminally is inactivated by 90% or more when incubated with 10 mmol/1DTE for 2.5 hours at 37° C., induces an activity for alkalinephosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l inthe presence of suramin, is not modified by cholesterol.
 2. Process forthe production of a post-translationally processed hedgehog proteinmutant by expressing a gene which codes for a hedgehog protein in abaculovirus expression system in a fermentation for a period of 24 to 27hours, purifying the cell supernatant in the presence of a proteaseinhibitor and a non-ionic detergent and isolating the hh mutant whichbinds to heparin-Sepharose and hydroxylapatite and is characterized inthat this hh mutant exhibits a molecular weight of 22+1 kDa underalkylating conditions, exhibits a molecular weight of 24+1 kD underreducing conditions, is stabilized with respect to its activity bysuramin is inactivated when 8 or more amino acids are cleavedN-terminally is inactivated by 90% or more when incubated with 10 mmol/lDTE for 2.5 hours at 37° C., induces an activity for alkalinephosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l inthe presence of suramin, is not modified by cholesterol.
 3. Process asclaimed in claim 2, wherein, after chromatography on heparin-Sepharose,it is dialysed against lower ionic strengths.
 4. Process as claimed inclaim 3, wherein the dialysis is carried out in the presence of 10-100mmol/l sodium chloride.
 5. Pharmaceutical composition containing a hhmutant as claimed in claim
 1. 6. Pharmaceutical composition as claimedin claim 5, containing suramin, a biocompatible matrix and/or asequestering agent.
 7. Process for the production of a pharmaceuticalcomposition by combination of a hh mutant as claimed in claim 1 with apharmaceutical auxiliary substance or with suramin.
 8. Process for theproduction of a pharmaceutical composition by combination of a hh mutantas claimed in claim 1 with a biocompatible matrix and/or a sequesteringagent.